Keywords

1 Introduction

Paddy is one of the main agricultural commodities in Indonesia. As a result, there are so many kinds of rice cultivar cultivated in Indonesia. Paddy ‘ Cempo Merah ’ is one of them. It is a pigmented local rice cultivar which is frequently cultivated by Indonesian farmers . Compared to the non-pigmented rice and black rice , red rice has higher phenolic compounds [1].

The growth and development of paddy is greatly determined by the nutrients availability in the soil such as copper (Cu). Copper is used by paddy plants as the co-factor on the enzymatic and non-enzymatic protein biosynthesized in their metabolic system. Copper is a co-factor of enzymatic protein such as cyt-c-oxidase and plastocyanin which are very important in photosynthetic process. Copper has an important role in carbohydrate and protein metabolism, polyphenol oxidases formation , and involves in Fe mobilization of plants [2]. The high concentration of copper in the soil can cause reactive oxygen species (ROS) to be formed, which then will lead oxidative stress on the plants [3]. The paddy’s productivity is greatly affected by the addition of copper in the soils. Paddy’s productivity declined into 10%, 50%, and 90% as the result of the addition 100mg/L, 300mg/L, and 1000mg/L of copper in the soils [2]. Copper accumulation in the soils and plant organs has been known as one reason of decreasing in the paddy’s productivity because it causes oxidative stress and leads to the death cells of plants organs [4]. Copper can induce ROS formation via Haber-Weiss reaction [5]. The plants’ response to the oxidative stress will be in the form of a collaborative enzymatic response, i.e. an anti-oxidative enzyme activity such as SOD activity and also a non-enzymatic response such as phenolic compounds production [6]. This study was carried out in order to study the effects of Cu2+ treated water on plant growth, its accumulation, superoxide dismutase (SOD ) activity and total phenolic compounds of paddy (Oryza sativa L. ‘Cempo Merah’) leaves.

2 Methods

2.1 Experimental Site

Paddy (Oryza sativa L. ‘Cempo Merah’) seeds used in this study were obtained from Berkat Agro Jaya Godean. The greenhouse experiment was carried out in Bambanglipuro, Bantul, D.I. Yogyakarta . The soil used for experiment was alluvial soil from agricultural soil. Each pot of 8kg growth medium (6kg of soils and 2kg of compost fertilizers) was cultivated with 21 days seedling and water to the moist level of 70% to 80% of field capacity. ZA fertilizers were applied to the plants’ growth medium 14 days post-cultivated. Copper at concentrations of 0mg/L (control), 100mg/L, and 300mg/L was added to the plants 28 days post-cultivated. CuSO4 solution and Ba(OH)2 solution was mixed in order to obtain the pure of Cu2+ solutions.

2.2 Sampling and Chemical Analysis

Plant and soil samples were taken four times, i.e. (0; 4; 72; and 168) h post-Cu treated. Plant samples (roots, stem, and leaves) were collected and washed with water. The monitoring indicators for plants growth included plants’ height, the number of plants’ leaves and tillers, biomass, and pH of soil. The plants’ height, the number of leaves and tillers, and pH of soil were being observed every week and at the day of sampling. The total copper in soil and leaves were determined by flame-AAS (Atomic Absorption Spectroscopy) in Laboratorium Penelitian dan Pengujian Terpadu (LPPT) Universitas Gadjah Mada.

2.3 SOD Activity Analysis

The paddy leaves (0.25g) were homogenized with 0.1M phosphate buffer (pH7.8) and 0.1mM EDTA, and centrifuged two times (13,000rpm for 10min). All steps of the extraction procedure were carried out at 0°C to 5°C. The superoxide dismutase (SOD) activity of paddy (Oryza sativa L. ‘Cempo Merah’) leaves was determined by using Giannopolitis & Ries method. The reaction mixture contain enzyme extracts; 1.3mM riboflavin; 13mM L-methionine; 63μM nitro-blue-tetrazolium (NBT); 0.05M carbonate buffer (pH10.2) and aquadest. Cuvette test tubes containing the mixture were illuminated with a fluorescent lamp 60W with 25cm in distance for 30s. Identical tubes which were not illuminated served as blanks. The absorbance was measured at 560nm. The illumination steps until absorbance measurement were repeated five times. Bovine Serum Albumin (BSA) was used as standard solutions.

2.4 Total Phenolic Compounds (TPC) Analysis

Total phenolic compounds of paddy (Oryza sativa L. ‘Cempo Merah’) leaves was determined by using Folin-Ciocalteau method. The paddy leaves (25mg) were extracted and homogenized with 0.1M phosphate buffer (pH6.5); centrifuged two times (10,000rpm for 15min) and added with ethanol (p.a). The liquid extract were mixed with Folin-Ciocalteu reagent (1:1); Na2CO3 7,5%, aquadest, and being incubated for about 1 hour. The sample’s absorbance was measured at 650nm and gallic acic was used as standard solutions.

3 Result and Discussion

The results of the effect of copper treatments (at 28 days post-cultivation) on plant growth of Oryza sativa L. ‘Cempo Merah’ plants can be seen in Table 1. Copper is one of important micronutrients for plant growth but in higher dose, copper may cause metabolic disorder and growth inhibition for plant [3]. The data in Table 1 show the effects of copper application on plant growth of Oryza sativa L. ‘Cempo Merah’. The height of plant reaches its highest value of 68cm at control treatment (35 days post-cultivation). The application of 100mg/L and 300mg/L copper in the water induces the decreasing of Oryza sativa L. ‘Cempo Merah’ height. The plants’ height significantly drop to 58.33cm at the treatment of 300mg/L copper.

Table 1 Effects of added copper on plants’ height, number of leaves, and number of tillers
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The effects of copper application on number of leaves and tillers were the same way with plants’ height. The number of plants’ leaves and tillers of paddy ‘Cempo Merah’ is decreasing the higher the number of copper concentration being added. The effects of copper application on plant growth also can be observed from plants’ biomass (Table 2).

Table 2 Effects of added copper on plants’ biomass
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Plants’ biomass of Oryza sativa L. ‘Cempo Merah’ drops significantly along with the increasing number of copper concentrations. The fresh and dry weight of root, stem, and leaf of Oryza sativa L. ‘Cempo Merah’ with the treatments of 100mg/L and 300mg/L of copper are lower than control treatment. These treatment is greatly affected to the fresh and dry weight of root organ, followed by leaf and then stem. It can be caused by the accumulation of copper in the soil and root which lead to the death of root cells. The reduction in the number of plants’s leaves, tillers, plants’ height, and plants’ biomass of paddy plant indicates that there is growth inhibition. The growth inhibition can be influenced by the copper toxicity. Copper toxicity can lead to sub-cellular disorder, oxidative stress [3], Fe deficiency [7], and the photoreduction inhibition [8]. The deficiency of Fe and sub-cellular disorder will induce the reduction of photosyntate productions . Therefore, the photosynthate allocations in the plants’ organs will not be enough for plant growth [9].

The effects of copper application on Cu accumulation in the soil of growth medium of Oryza sativa L. ‘Cempo Merah’ can be seen in Table 3. Copper concentration in the soil is significantly increased the higher the number of copper concentrations added in the soil. The highest copper concentration in the soil is found in the soil treated by 300mg/L copper at the interval period of 4 h post-copper application. Plants usually uptake copper from the soil in the form of copper ions (Cu2+). The reduction of copper concentration in the soil at time periods of 72 h and 168 h post-copper application indicate the copper uptake by paddy plants.

Table 3 Effects of added copper on copper concentration in soil
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The rate of copper uptake by plant can also be observed from the copper accumulation in the plant tissues. In this study, the copper accumulation in the Oryza sativa L. ‘Cempo Merah’ can be seen in Table 4. Copper accumulation in paddy leaves reflects the copper concentration in the soil. The copper concentration in the paddy leaves increase upon the higher the number of copper concentration in the soil.

Table 4 Effect of added copper on copper accumulation in plant leaves
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The rate of copper uptake of plant from the soil are greatly influenced by soil composition, copper concentration in the soil, bioavailability of plant, micronutrients antagonistics [10], storage capacity of vacuoles [11], and soil pH [12]. The soil pH of this study are approximately 6.4 to 6.42. The copper solubility and mobility will be increased at the soil pH higher than 7 [12]. Therefore, this soil pH conditions can cause the increasing number of copper solubility, copper mobility, and copper uptake by paddy plant.

The effects of copper application on superoxide dismutase activity of paddy leaves in this study can be seen in the Table 5. Copper application in irrigation water at time period of 28 days post-cultivated can increase the superoxide (SOD) activity in the Oryza sativa L. ‘Cempo Merah’ leaves. The highest SOD activity in paddy leaves is shown in 300mg/L copper treatment, i.e. 7.83 unit per gram of paddy leaves at time interval of 4 h post application.

Table 5 Effects of added copper on superoxide dismutase activity of paddy leaves
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The increasing of SOD activity in paddy ‘Cempo Merah’ leaves by the application of copper can be the result of Cu-Zn SOD activation induced by reactive oxygen species (ROS) formation through Haber-Weiss reaction [5]. As a first defense enzyme, SOD has an important role to fight against ROS. ROS formation can lead oxidative stress for organism [13]. Yet, the reduction activity of SOD at time interval 72 h and 168 h post-copper application indicates that there is another anti-oxidative mechanism. Plant has several anti-oxidative responses in order to overcome oxidative stress , i.e. enzymatic and non-enzymatic responses [13].

Phenolic compounds production is one of non-enzymatic anti-oxidative responses which is regulated by plant to overcome oxidative stress. Phenolic compounds are produced by plant via shikimate pathways. The effect of copper application in the irrigation water on the total phenolic compounds of Oryza sativa L. ‘Cempo Merah’ can be seen in Table 6.

Table 6 Effects of added copper on total phenolic compounds of paddy leaves
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The effect of copper application on total phenolic compounds is the same way with the effect on the SOD activity of paddy ‘Cempo Merah’ leaves. Total phenolic compounds of paddy leaves increases upon the increasing of copper concentration treatments. The highest total phenolic compounds of paddy leaves is shown by the treatment of copper with the concentration 300mg/L at interval time 4 h post-copper application, i.e. 7.83 unit per gram of leaves.

Phenolic compounds inhibits the ROS formation through strong binding with pro-oxidant of Cu. The increasing in the number of copper concentrations will stimulate the phenolic compounds production [14]. Therefore, the addition of copper concentrations in this study can cause the total phenolic compounds in the paddy leaves to increase. In addition, the increasing of total phenolic compounds of paddy leaves in this study can be the result of peroxidases activity.

4 Conclusions

Copper application significantly induces the growth inhibition, accumulation of copper in the soil and leaves, the increasing of superoxide dismutase activity and total phenolic compounds production of Oryza sativa L. ‘Cempo Merah’ leaves.